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. 2011 Feb 1;108(5):2130-5.
doi: 10.1073/pnas.1009933108. Epub 2011 Jan 18.

Human papillomavirus E7 oncoprotein induces KDM6A and KDM6B histone demethylase expression and causes epigenetic reprogramming

Margaret E McLaughlin-Drubin  1 , Christopher P Crum, Karl Münger
Affiliations

Human papillomavirus E7 oncoprotein induces KDM6A and KDM6B histone demethylase expression and causes epigenetic reprogramming

Margaret E McLaughlin-Drubin et al. Proc Natl Acad Sci U S A. .

Abstract

Despite the availability of vaccines, human papillomavirus (HPV) infections remain a cause of significant cancer morbidity and mortality. We have previously shown that HPV16 E7 associates with and diminishes E2F6-containing polycomb repressive complexes. Here, we show that repressive trimethyl marks on lysine 27 of histone 3, which are necessary for binding of polycomb repressive complexes, are decreased in HPV16 E7-expressing cells and HPV16-positive cervical lesions. This is caused by transcriptional induction of the KDM6A and KDM6B histone 3 lysine 27-specific demethylases. HPV16 E7-mediated KDM6B induction accounts for expression of the cervical cancer biomarker, p16(INK4A). Moreover, KDM6A- and KDM6B-responsive Homeobox genes are expressed at significantly higher levels, suggesting that HPV16 E7 results in reprogramming of host epithelial cells. These effects are independent of the ability of E7 to inhibit the retinoblastoma tumor suppressor protein. Most importantly, these effects are reversed when E7 expression is silenced, indicating that this pathway may have prognostic and/or therapeutic significance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The repressive H3K27me3 mark is specifically reduced in HPV16 E7-expressing primary human epithelial cells. (A) Immunofluorescence analysis of histone H3 methylation in primary human epithelial cells (HFKs) and donor- and passage-matched HPV16 E7-expressing HFKs (HFK/E7). (B) Western blot analysis of H3K27me3 levels in HFKs and HFK/E7s. Lysates were separated by SDS/PAGE, transferred, and probed for H3K27me3 (me3) and total H3 as a loading control.
Fig. 2.
Fig. 2.
Increased expression of KDM6A and KDM6B in HPV16 E7-expressing primary human epithelial cells. (A) Immunofluorescence analysis of KDM6B (Upper) and KDM6A (Lower) expression in donor- and passage-matched populations of primary human foreskin keratinocytes (HFKs; Left) and HPV16 E7-expressing HFKs (HFK/E7; Right). (B) Western blot analysis of KDM6A and KDM6B levels in HFKs and HFK/E7 cells. Lysates were separated by SDS/PAGE, transferred, and probed for KDM6A, KDM6B, and HPV16 E7. A pRB blot is shown to document functional E7 expression, and an actin blot is included as a loading control. (C) Quantitative real-time RT-PCR analysis of KDM6A and KDM6B mRNA expression in HFK and HFK/E7 cells. The bar graph shows averages and SDs from three independent experiments, each performed in triplicate. Increases in HFK E7 cells are statistically significant (*), with P values < 0.005.
Fig. 3.
Fig. 3.
HPV16 E7-mediated induction of KDM6B is critical for the expression of the cervical cancer biomarker p16INK4A. (A) Coimmunofluorescence staining of p16INK4A and H3K27me3 in an HPV16-positive cervical intraepithelial neoplasia (CIN) specimen. Upper and Lower are from different areas of the same specimen. Similar staining patterns were detected in three additional CIN specimens. Hoechst staining is shown to visualize nuclei, and a phase picture shows cellular morphology. (B) Monolayer cultures of HPV16-immortalized HFKs were transfected with KDM6B-specific siRNA duplexes or control siRNA (CTL), and p16INK4A and p14ARF expression was analyzed by Western blotting at 72 h posttransfection. An actin blot is shown as a loading control (Left). Averages and SDs of KDM6B, p16INK4A, and p14ARF levels from three independent experiments are shown on the bar graph in Right. Statistically significant decreases (P < 0.05) are indicated by an asterisk.
Fig. 4.
Fig. 4.
HPV16 E7-mediated induction of KDM6B and its transcriptional target p16INK4A are not dependent on the ability of HPV16 E7 to degrade pRB. Western blot analysis of KDM6B and p16INK4A expression in donor- and passage-matched populations of primary human fibroblasts (HFFs) expressing WT HPV16 E7, the pRB binding/degradation-deficient HPV16 delD21-24 mutant, and control vector-infected HFFs. An E7 blot shows similar expression of WT and mutant E7, and an actin blot is show as a loading control.
Fig. 5.
Fig. 5.
Dysregulation of HOX gene expression in HPV16 E7-expressing primary human epithelial cells. Quantitative real-time RT-PCR of HOX mRNA expression in primary human foreskin keratinocytes (HFKs) and donor- and passage-matched HPV16 E7-expressing HFKs (HFK/E7). Bar graphs represent averages and SDs of three experiments, each performed in triplicate. HOX genes that exhibit significant (P < 0.05) up-regulation are marked (*). Although all 39 genes in the HOX A–D clusters were analyzed, those HOX genes that were expressed in HFKs at levels too low to evaluate are not shown.
Fig. 6.
Fig. 6.
HPV16 E7-mediated induction of KDM6B and p16INK4A and decreases in H3K27me3 levels are associated with decreased H3K37 trimethylation and increased KDM6B binding to the INK4A promoter and are reversible. (A) Western blot analysis of KDM6B, p16INK4A, and HPV16 E7 expression in U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7 treated with doxycycline as indicated. Lysates were separated by SDS/PAGE, transferred, and probed with the indicated antibodies. An actin blot is shown as a loading control. (B) Immunofluorescence analysis of histone H3 lysine 27 trimethylation in U2OS-tet on cells with doxycycline-inducible expression of HPV16 E7 treated with doxycycline as indicated. Hoechst stain is shown to visualize nuclei. (C and D) ChIP assays using lysates from U2OS-tet on cells with or without doxycycline-inducible expression of HPV16 E7. In C, an antibody specific for H3K27me3 was used, and in D, an antibody specific to KDM6B was used. qPCR was used to measure the degree of enrichment. The H3K27me3 signal was normalized to histone H3 bound, and the KDM6B results are presented as a percentage of bound/input. Statistically significant changes (P < 0.05) are indicated by an asterisk.
Fig. 7.
Fig. 7.
KDM6A and KDM6B depletion causes growth suppression in a cervical carcinoma line. KDM6A or KDM6B were depleted in the HPV16-positive CaSki cervical carcinoma cells followed by colony formation assays. Averages and SDs for three independent experiments are shown. Statistically significant changes (P < 0.05) are indicated by an asterisk.
Fig. 8.
Fig. 8.
Model for epigenetic reprogramming by the HPV16 E7 oncoprotein. Details are in the text.
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